The long term objective of this proposal is determination of the molecular details by which the drugs of the digitalis class (cardiac glycosides) interact with their pharmacological receptor, the (Na,K)-ATPase. To understand this process, the structure of the receptor and the drug, and the effect of changes in these structures on the stability (affinity) of the drug-receptor complex must be determined. Human heart contains three isoforms, alpha1 being predominant in muscle and alpha2 and alpha3 being enriched in the conducting system. If the alpha1 isoform is primarily responsible for the increase in contractility and alpha2 and/or alpha3 primarily responsible for the toxic effect, a cardiac glycoside having a relatively higher affinity for alpha1 (and therefore possessing an improved therapeutic index) might be discovered or synthesized. Specific Aim 1 is to characterize the three alpha isozymes of human (Na,K)-ATPase expressed in NIH 3T3 cells with respect to their differential interaction with a series of cardiac glycosides. Specific Aim 2 is to identify and locate within the primary sequence, amino acid residues in the (Na,K)- ATPase that are involved in interacting with cardiac glycosides. The sites of interest include ligand regulatory sites (Mg, ATP, Na and K) as well as the actual cardiac glycoside binding site. The structure of (Na,K)-ATPase will be altered using site-directed mutagenesis of sheep alpha1 isoform expressed in NIH 3T3 or in Hela cells. The structure of the drug will be varied by taking advantage of existing digitalis analogous. For Aims 1 and 2 the dissociation constant, KD, will be determined by saturation binding assays or by measurement of kinetic rate constants or alternatively, deduced from inhibition studies (150). The alteration in KD for binding of a series of cardiac glycosides to each mutant will define the role specific amino acids mutated play in the binding process. Specific Aim 3 is to obtain and characterize preparations of the alpha-beta complex, separated alpha and beta subunits and selected smaller protein fragments of sheep (Na,K)-ATPase of appropriate purity and in sufficient amounts for three dimensional structural studies. Purity will be assessed using native and denaturing gels and Western blots developed using antibodies against the sheep alpha and beta subunits. Degree of glycosylation, number and intactness of disulfide bonds and secondary structure (circular dichroism and infrared spectroscopy) will be determined. A working laboratory model of the receptor consistent with the experimental data will be constructed using techniques of energy minimization. The proteins, when pure, will be submitted for crystallization trials.